Method of transforming soybean

ABSTRACT

The present disclosure provides methods for  Agrobacterium -mediated transformation of soybean cells or tissue and regeneration of the transformed cells or tissue into transformed plants. The methods may be used for transforming many soybean cultivars.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/390,562, filed Jun. 22, 2002, the entire contents of which ishereby incorporated by reference.

FIELD OF THE INVENTION

The invention relates generally to methods for plant transformation and,more particularly, to methods for transforming soybean cells or tissues.The invention also relates to methods for regenerating transgenicsoybean plants from transformed soybean cells or tissues. The inventionalso relates to transgenic soybean plants and seeds obtained by suchmethods.

BACKGROUND

Soybean is a major food and feed source that is grown on more acresworldwide than any other dicotyledonous crop. It is reportedly grown onmore than 50 million hectares. Unfortunately, only a few plantintroductions have given rise to the major cultivars grown in the UnitedStates and, as a consequence, this narrow germplasm base has limitedsoybean breeding potential. The limited genetic base in domestic soybeanvarieties has limited the power of traditional breeding methods todevelop varieties with improved or value-added traits.

Hence, the use of genetic engineering techniques to modify soybean canfacilitate the development of new varieties with, for example, traitssuch as herbicide resistance, disease resistance (such as virusresistance, for example), and seed quality improvement in a manner thathas been unattainable by traditional breeding methods or tissue-cultureinduced variation.

The development of an efficient transformation system is necessary forthe analysis of gene expression in plants. The requirements for such asystem include a proper target plant tissue that will allow efficientplant regeneration, a gene delivery vehicle that delivers foreign DNAefficiently into the target plant cells, and an effective method forselecting transformed cells. In genetic transformation of dicotyledonousspecics, transformation systems utilizing the bacterium Agrobacteriumtumefaciens have been frequently used as vehicles for gene delivery. Thepreferred target tissues for Agrobacterium-mediated transformationpresently include cotyledons, leaf tissues, and hypocotyls. Highvelocity microprojectile bombardment offers an alternative method forgene delivery into dicotyledonous plants.

Agrobacterium-mediated gene delivery in soybean has been far fromroutine. In reports that have been available to the public, meristemsand cotyledon tissues have been frequently mentioned as targets for usein Agrobacterium-mediated gene delivery. However, reliable and efficienttransformation and regeneration from these two explant sources are oftennot accomplished.

U.S. Pat. No. 5,169,770 and 5,376,543 to Chee et al. discuss anon-tissue culture method of transforming soybeans to produce transgenicplants, wherein seeds are germinated and meristematic or mesocotyl celltissues are inoculated with bacterial cells, specifically Agrobacteriumstrains, which, through infection, transfer DNA into the explants. Thismethod depends on the growth of preformed shoots.

Parrott W. A. et al. (1989), “Recovery of primary transformants ofsoybean,” Plant Cell Reports 7:615-617, report recovery of soybeantransformants from immature cotyledon tissue after co-cultivation withAgrobacterium. However, the regenerated plants were chimeric, and thetransgenes were not transmitted to the progeny.

U.S. Pat. No. 5,416,011 (to Hinchee et al.) discusses utilizing acotyledon explant, which requires removal of the hypocotyl, saving andseparating the cotyledons and inserting a chimeric gene by inoculationwith Agrobacterium tumefaciens vectors containing the desired gene.

Yan B. et al. (2000), “Agrobacterium tumefaciens—mediated transformationof soybean using immature zygotic cotyledon explants,” Plant CellReports 19:1090-1097, report an overall 0.03% transformation frequencyin Agrobacterium-mediated transformation in soybean with immaturecotyledons.

U.S. Pat. No. 6,384,301 to Martinell et al. describesAgrobacterium-mediated gene delivery into cells in the meristem of anisolated soybean embryonic axis. Their method does not involve acallus-phase tissue culture.

From the work described above, it is clear that the goal of establishinga reliable soybean transformation system is seldom accomplished by theworkers involved when meristems and cotyledon tissues are used as sourceexplants for Agrobacterium-mediated gene delivery. Therefore, there is aneed to continue to exploit new methodology, including new sourceexplants, in order to develop a more efficient soybean transformationsystem.

It has been demonstrated in soybean tissue culture that plantregeneration may be achieved from epicotyl tissues and primary leaftissues. However, to-date, no successful transformation has beenreported in soybean using these two explant sources as targets for genedelivery.

Wright M. S. et al. (1987) “Initiation and propagation of Glycine max L.Merr.: Plants from tissue-cultured epicotyls,” Plant Cell Reports8:83-90, describes successful initiation and proliferation of shootsfrom epicotyl tissue of soybean. Explanted epicotyls were induced toform shoots in Schenk and Hildebrandt medium containing 20 μM kinetinfor 5 weeks. Shoot proliferation was maintained on N6 medium containing2.1 nM picloram and 0.1 μM benzyladenine.

Wright M. S. et al. (1987) “Regeneration of soybean (Glycine max L.Merr.) from cultured primary leaf tissue,” Plant Cell Reports 6:83-89,describes a reproducible method for regeneration of plants from primaryleaf tissue of 27 varieties of soybean They found that while2,4,5-trichlorophenoxyacetic acid was demonstrated to be essential forregeneration, addition of benzyadenine (BA) was found to enhanceregeneration.

Rajasckaren K. et al. (1997) “Somatic embryogenesis from culturedepicotyls and primary leaves of soybean (Glycine max L. Merr),” In VitroCellular & Developmental Biology 33(2):88-91, describes regeneration ofseveral varieties of soybean by somatic embryogenesis from culturedepicotyls and primary leaf tissues of immature seeds from greenhousegrown plants. They found that somatic embryogenesis was induced fromepicotyls and primary leaves when cotyledon halves with the intactzygotic embryo axes were cultured on Murashige and Skoog (MS) mediumsupplemented with 46.2 μM 2,4-D. In the absence of being cultured withthe cotyledon halves, no embryogenesis was observed from isolated axes,epicotyls or primary leaves. Rapid multiplication of shoot tips fromgerminating somatic embryos was achieved on Cheng's basal mediumcontaining 11.3 μM benzyladenine.

SUMMARY

The present invention provides a method for transforming soybean cellsand regeneration of the transformed cells into transformed plants. Themethod may be used for transforming many soybean cultivars.

The invention provides a novel soybean explant that enablesAgrobacterium tumefaciens-mediated gene delivery into soybean cells withhigh efficiency.

In particular, the invention provides a method for transforming soybeancells or tissue, the method comprising:

-   -   (a) preparing an explant from a soybean seed by:        -   (i) removing all or a part of the hypocotyl from said seed;        -   (ii) removing one cotyledon along with its adjacent axillary            bud from the seed, and leaving one cotyledon with the            epicotyl and primary leaves attached thereto;        -   (iii) removing a portion of a primary leaf from the            remaining cotyledon, thereby generating a primary leaf base;            and    -   (b) co-cultivating the explant with Agrobacterium comprising a        nucleic acid of interest to be incorporated into the genome of        the soybean cells.

In additional embodiments, the method further includes one or more ofthe following: inducing shoot formation from the primary leaf base andthe adjacent epicotyl; cultivating the shoot in a medium containing aselection agent; selecting a transformed shoot; and regenerating atransformed plant from the transformed shoot.

In a further embodiment, the invention provides a method for producing astably transformed soybean plant, the method comprising:

-   -   (a) preparing an explant from a soybean seed by:        -   (i) removing all or a part of the hypocotyl from said seed;        -   (ii) removing one cotyledon along with its adjacent axillary            bud from the seed, and leaving one cotyledon with the            epicotyl and primary leaves attached thereto;        -   (ii) removing a portion of a primary leaf from the epicotyl,            thereby generating at least one primary leaf base;    -   (b) co-cultivating the explant with Agrobacterium comprising a        nucleic acid of interest to be incorporated into the genome of        the soybean cells;    -   (c) inducing shoot formation from the primary leaf base area;    -   (d) cultivating a formed shoot in a medium containing a        selection agent,;    -   (e) selecting a transformed shoot; and    -   (f) regenerating a selected transformed shoot into a soybean        plant.

In another embodiment, a portion of each of the primary leaves of theexplant generated in (a)(ii) is removed, thereby generating a pair ofprimary leaf bases.

The method of the invention may be employed to introduce any desirednucleic acid into a soybean cell. In one embodiment of the invention,the nucleic acid comprises a gene that would express a desirableagronomic trait in soybean.

In another embodiment of the invention, the nucleic acid comprises aphosphomannose isomerase gene, which is used as a selectable markergene.

In an additional embodiment of the invention, the co-cultivating of theexplant with Agrobacterium is carried out in the presence of mannose.

Both mature and immature seeds may be employed to generate the explantused in the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a map of plasmid pNOV2105.

FIG. 2 shows a map of plasmid pNOV2145.

FIG. 3 shows a map of plasmid pNOV2147.

FIG. 4 shows an exemplary process for preparing a soybean explant. PanelA depicts a soybean seed embryo in which a part of the hypocotyl isremoved. Panel B depicts the soybean explant from Panel A in which onecotyledon is removed along with its adjacent axillary bud. Panel Cdepicts the soybean explant from Panel B after removal of the twoprimary leaves, generating a break point at the base of each primaryleaf.

FIG. 5 shows a map of plasmid pBSC11234.

FIG. 6 shows a map of plasmid pBSC11369.

DETAILED DESCRIPTION

The present invention will now be described more fully hereinafter withreference to the accompanying figures, in which various embodiments ofthe invention are described. This invention may, however, be embodied indifferent forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete and will fully conveythe scope of the invention to those skilled in the art. The terminologyused in the description of the invention herein is for the purpose ofdescribing particular embodiments only and is not intended to belimiting of the invention. As used in the description of the inventionand the appended claims, the singular forms “a”, “an” and “the” areintended to include the plural forms as well, unless the context clearlyindicates otherwise.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

Except as otherwise indicated, standard methods may be used for theproduction of cloned genes, expression cassettes, vectors (e.g.,plasmids), proteins and protein fragments, and transformed cells andplants according to the present invention. Except as otherwiseindicated, standard methods may be used for the production of clonedgenes, expression cassettes, vectors (e.g., plasmids), proteins andprotein fragments according to the present invention. Such techniquesare known to those skilled in the art. See e.g., J. Sambrook et al.,Molecular Cloning: A Laboratory Manual Second Edition (Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1989), and F. M. Ausubel etal., Current Protocols In Molecular Biology (Green PublishingAssociates, Inc. and Wiley-Interscience, New York, 1991); J. Draper etal., eds., Plant Genetic Transformation And Gene Expression: ALaboratory Manual, (Blackwell Scientific Publications, 1988); and S. B.Gelvin & R. A. Schilperoort, eds., Introduction, Expression, AndAnalysis Of Gene Production In Plants.

The present invention is drawn to methods and compositions for thestable transformation of soybean with nucleic acid sequences of interestand the regeneration of transgenic soybean plants.

The methods of the invention may be employed to express any nucleic acidof interest in soybean plants. A gene of interest may be, for example, agene for herbicide resistance, disease resistance, or insect/pestresistance, or is a selectable or scorable marker, and comprises aplant-operable promoter, a coding region, and a 3′ terminator region.Herbicide resistance genes include the AHAS gene for resistance toimidazolinone or sulfonyl urea herbicides, the pat or bar gene forresistance to bialaphos or glufosinate, the EPSP synthase gene forresistance to glyphosate, etc. Disease resistance genes include genesfor antibiotic synthetic enzymes, e.g., for pyrrolnitrin syntheticenzymes, plant derived resistance genes, and the like. Insect resistancegenes include genes for insecticidal proteins from Bacillusthuringiensis. Genes of interest may also encode enzymes involved inbiochemical pathways, the expression of which alters a trait that isimportant in food, feed, nutraceutical, and/or pharmaceuticalproduction. The gene of interest may be located on a plasmid. A plasmidsuitable for use in the present invention may comprise more than onegene of interest and/or the Agrobacterium may comprise differentplasmids having different genes of interest.

The present invention provides a method for the transformation ofvarieties of soybean, including Glycine max. The method is based onAgrobacterium-mediated delivery of a desired gene into a soybean cellfollowed by regeneration of transformed cell(s) into a transformedsoybean plant. The methods of the invention are cultivar independent.

In one embodiment of the invention, an explant is prepared bygerminating a soybean mature seed or immature seed collected from agreenhouse grown plant in a seed germination medium for a period oftime, removing seed coat and, subsequently, a cotyledon from said matureseed or immature seed. In a preferred embodiment of the invention, aportion of the exposed primary leaves is then removed, thereby creatinga break point at the primary leaf base (FIG. 4). Agrobacterium-mediatedgene delivery is made into the cells at the primary leaf base or in thearea of the primary leaf break point. Adventitious shoots are inducedfrom the primary leaf base area of the epicotyl. This induction isachieved by removing pre-existing meristems (i.e., primary, secondary,and axillary meristems) and subjecting the explant to a shoot inductionmedium containing appropriate growth regulators, The shoot inductionprocess facilitates the development or regeneration of transformedshoots from the targeted primary leaf base cells.

Transformed soybean cells are cultured in the presence of a selectionagent. Preferably, the cells are transformed with a phosphomannoseisomerase (PMI gene, and the transformed cells are cultivated in thepresence of mannose. In a medium that contains mannose as a selectionagent, soybean cells transformed with a PMI gene have a growth advantageover those that are not so transformed.

The time required for regenerating a transformed soybean plant using themethod described in this invention is significantly reduced compared toother Agrobacterium-mediated transformation protocols that are reportedin the literature. A rooted transformed soybean shoot may be produced 8to 12 weeks from the initiation of a transformation experiment. Aforeign genetic construct, or transgene, to be inserted into the soybeangenome is created in vitro by normal techniques of recombinant DNAmanipulations. The construct may be comprised of any heterologousnucleic acid. The genetic construct is transformed into theAgrobacterium strain for delivery into the soybean cells. TheAgrobacterium is non-oneogenic, and several such strains are now widelyavailable. The Agrobacterium is preferably selected from A. tumefaciensand A. rhizogenes.

The foreign genetic construct preferably comprises a selectable markergene. The preferred selectable marker gene is a phosphomannose isomerasegene. Other suitable selectable marker genes include, but are notlimited to, genes encoding: neomycin. phosphotransferase II (Fraley etal., CRC Critical Reviews in Plant Science 4, 1 (1986)); cyanamidehydratase (Maier-Greiner et al., Proc. Natl. Acad. Sci. USA 88, 4250(1991)); aspartate kinase; dihydrodipicolinate synthase (Perl et al.,BioTechnology 11, 715 (1993)); bar gene (Toki et al., Plant Physiol.100, 1503 (1992); Meagher et al., Crop Sci. 36, 1367 (1996));tryptophane decarboxylase (Goddijn et al., Plant Mol. Biol. 22, 907(1993)); neomycin phosphotransferase (NEO; Southern et al., J. Mol.Appl. Gen. 1, 327 (1982)); hygromycin phosphotransferase (HPT or HYG;Shimizu et al., Mol. Cell. Biol. 6, 1074 (1986)); dihydrofolatereductase (DHFR); phosphinothricin acetyltransferase (DeBlock et al.,EMBO J. 6, 2513 (1987)); 2,2- dichloropropionic acid dehalogenase(Buchanan-Wollatron et al., J. Cell. Biochem. 13D, 330 (1989));acetohydroxyacid .synthase (U.S. Pat. No. 4,761,373 to Anderson et al.;Haughn et al., Mol. Gen. Genet. 221, 266 (1988));5-enolpyruvyl-shikimate-phosphate synthase (aroA; Comai et al., Nature317, 741 (1985)); haloaryinitrilase (WO 87/04181 to Stalker et al.),acetyl-coenzyme A carboxylase (Parker et al., Plant Physiol. 92, 1220(1990)); dihydropteroate synthase (sulI; Guerineau et al., Plant Mol.Biol. 15, 127 (1990)); and 32 kDa photosystem II polypeptide (psb A;Hirschberg et al., Science 222, 1346 (1983)).

Also included are genes encoding resistance to chloramphenicol(Herrera-Estrella et al., EMBO J. 2, 987 (1983)); methotrexate(Herrera-Estrella et al., Nature 303, 209 (1983); Meijer et al., PlantMol. Biol. 16, 807 (1991)); hygromycin (Waldron et al., Plant Mol. Biol.5, 103 (1985); Zhijian et al., Plant Science 108, 219 (1995); Meijer etal., Plant Mol. Bio. 16, 807 (1991)); streptomycin (Jones et al., Mol.Gen. Genet. 210, 86 (1987)); spectinomycin (Bretagne- Sagnard et al.,Transgenic Res. 5, 131 (1996)); bleomycin (Hille et al., Plant Mol.Biol. 7, 171 (1986)); sulfonamide (Guerineau et al., Plant Mol. Bio. 15,127 (1990); bromoxynil (Stalker et al., Science 242, 419 (1988)); 2,4-D(Streber et al., Biol/Technology 7, 811 (1989)); phosphinothricin(DeBlock et al., EMBO J. 6, 2513 (1987)); spectinomycin(Bretagne-Sagnard and Chupeau, Transgenic Research 5, 131 (1996)).

In one embodiment, the starting material for the transformation processis a soybean mature seed. In another embodiment, the starting materialcan be a soybean immature seed from a growing soybean plant. The seed isplaced on a germination medium and permitted to germinate for a periodof 6-24 hours, preferably for about 6-14 hours, and more preferably forabout 8-12 hours. Seeds may also be allowed to germinate for a longerperiod of time, for example, from 2 to 5 days, if desired.

The seed coat and hypocotyl of the germinating seed is removed. Onecotyledon along with its adjacent axillary shoot bud is also removed.Afterwards, the primary leaves are substantially removed, therebycreating an explant comprising the primary leaf base, epicotyl to whichthe leaf base is attached, and a cotyledon to which the epicotyl isattached. Substantially removed means removal of a major portion ofprimary leaf tissue.

For Agrobacterium-mediated gene transfer, wounding of the plant tissueis known to facilitate gene transfer. Therefore it is preferred, but notnecessary, that a wound is created at the leaf base region.

The explant, prepared as described above, is then immersed into anAgrobacterium cell suspension for a few minutes to a few hours,typically about 0.5-3 hours, and preferably 1-2 hours. ExcessiveAgrobacterium cell suspension is removed and the remaining Agrobacteriumare permitted to co-cultivate with the explant on a co-cultivationmedium for several days, typically two to five days, and preferablythree to four days, under 16h light/8h dark conditions at a temperatureof about 22° C.±2° C.

After co-cultivation, the explant is transferred to a medium (or aseries of media) conducive to shoot development and selection oftransformed cells, for 8-12 weeks. Such a medium (or media) generallycontains a shoot-inducing hormone as well as a selection agent. Theregeneration media used in the examples below contain mannose, as theselection agent, as well as benzylaminopurine (BAP), a shoot-inducinghormone. The term hormone also includes cell growth regulating compoundsthat induce shoot formation, including, but not limited to, auxins (suchas, e.g., IAA, NAA, and indole butyric acid (IBA)), cytokinins (such as,e.g., thidiazuron, kinetin, and isopentenyl adenine), and/or gibberellicacids (GA₃).

When shoots reach about 2 cm and with full trifoliate leaf formation,shoots are separated from the explant and placed on a rooting medium toinduce root formation. Preferably, the rooting medium also contains aselection agent to further help identify potential transformed shoots.Root formation takes approximately 1-2 weeks, following which the plantscan be transferred to soil and grown to full maturity.

Transgenic plants comprising a heterologous nucleic acid (i.e.,comprising cells or tissues transformed in accordance with the methodsdescribed herein), as well as the seeds and progeny produced by thetransgenic plants, are an additional aspect of the present invention.Procedures for cultivating transformed cells to useful cultivars areknown to those skilled in the art. Techniques are known for the in vitroculture of plant tissue, and in a number of cases, for regeneration intowhole plants. A further aspect of the invention is transgenic planttissue, plants, or seeds containing the nucleic acids described above.In a preferred embodiment, transformed plants produced using the methodsdescribed herein are not chimeric, or only a small proportion oftransformed plants is chimeric. This is preferably achieved by extendingthe period of high cytokinin treatment or by increasing the stringencyof mannose selection, or both.

Thus, the transformed cells of the present invention, identified byselection or screening and cultured in an appropriate medium thatsupports regeneration as provided herein, may then be allowed to matureinto plants. Plants are preferably matured either in a growth chamber orgreenhouse. Plants are regenerated from about 2-6 weeks after atransformant is identified, depending on the initial tissue. Duringregeneration, cells may be grown on solid media in tissue culturevessels. Illustrative embodiments of such vessels are petri dishes andPlant Con®s. After the regenerating plants have reached the stage ofshoot and root development, they may be transferred to a greenhouse forfurther growth and testing. As provided above, seeds and progeny plantsof the regenerated plants are an aspect of the present invention.Accordingly, the term “seeds” is meant to encompass seeds of thetransformed plant, as well as seeds produced from the progeny of thetransformed plants. Plants of the present invention include not only thetransformed and regenerated plants, but also progeny of transformed andregenerated plants produced by the methods described herein.

Plants produced by the described methods may be screened for successfultransformation by standard methods described above. Seeds and progenyplants of regenerated plants of the present invention may becontinuously screened and selected for the continued presence of thetransgenic and integrated nucleic acid sequence in order to developimproved plant and seed lines, which are another aspect of the presentinvention. Desirable transgenic nucleic acid sequences may thus be moved(i.e., introgressed or inbred) into other genetic lines such as certainelite or commercially valuable lines or varieties. Methods ofintrogressing desirable nucleic acid sequences into genetic plant linesmay be carried out by a variety of techniques known in the art,including by classical breeding, protoplast fusion, nuclear transfer andchromosome transfer. Breeding approaches and techniques are known in theart, and are set forth in, for example, J. R. Welsh, Fundamentals ofPlant Genetics and Breeding (John Wiley and Sons, New York, (1981));Crop Breeding (1). R. Wood, ed., American Society of Agronomy, Madison,Wis., (1983)); O. Mayo, The Theory of Plant Breeding, Second Edition(Clarendon Press, Oxford, England (1987)); and Wricke and Weber,Quantitative Genetics and Selection Plant Breeding (Walter de Gruyterand Co., Berlin (1986)). Using these and other techniques in the art,transgenic plants and inbred lines obtained according to the presentinvention may be used to produce commercially valuable hybrid plants andcrops, which hybrids are also an aspect of the present invention.

The foregoing is illustrative of the various embodiments of the presentinvention and is not to be construed as limiting thereof.

The invention will be further described by the following examples, whichare not intended to limit the scope of the invention in any manner.

EXAMPLE 1

Transformation Vectors

The plasmid pNOV2105 (FIG. 1) is a modification of p Victor, which isdisclosed and described in WO 97/04112 in that the 35S promoter isreplaced with a SMAS promoter, the 35S terminator is replaced with theNos terminator, and an additional SMAS promoter is inserted upstream ofthe GUSintronGUS sequence, which is flanked on its 3′ end by a Nosterminator. pNOV2105 employed in the methods described herein does notcontain the multicloning site that is found in p Victor. However, it iswell within the skill in the art to add such a cloning site, if desired.

pNOV2105 (FIG. 1) is a vector for Agrobacterium-mediated planttransformation and contains the Ti right and left border sequences fromthe nopaline type pTiT37 plasmid (Yadav et al. 1982 Proc Natl Acad Sci79:6322-6326) flanking the genes phosphomannose isomerase (PM) andbeta-glucoronidase (GUS).

For replication and maintenance in E. coli, the plasmid contains theorigin of replication from the E. coli plasmid pUC19 (pUC19ori)(Yanish-Perron et al. 1985 Gene 33:103-119), and for replication andmaintenance in Agrobacterium tumefaciens the plasmid further containsthe origin of replication from the Pseudomonas plasmid pVS1 (pVSlori)(Itoh et al. 1984 Plasmid 11:206-220; Itoh and Haas 1985 Gene 36:27-36).For selection in E. coli and Agrobacterium tumefaciens, the plasmidcontains the spectinomycin/streptomycin resistance gene (spec/strep)from the transposon Tn7 encoding the enzyme3″(9)-O-nucleotidyltransferase (Fling et al. 1985 Nucleic Acids Res19:7095-7106). The spec/strep resistance gene is fused to the tacpromoter (see, e.g., Amann et al. 1983 Gene 25(203):167-78) forefficient expression in the bacterium.

The T-DNA segment between the right and left border harbors thefollowing genes, which are the only genes transferred to the soybeanplant via the Agrobacterium tumefaciens-mediated transformation.

GUSintronGUS

beta-glucuronidase (GUS): This segment next to the right border containsthe beta-glucuronidase gene (GUS) from E. coli with an intron in thecoding region to prevent translation by Agrobacterium fused to the SMASpromoter and Nos terminator. The GUSintronGUS gene was isolated fromplasmid pBISN1. (Narasimhulu et al. 1986 Early transcription ofAgrobacterium DNA in tobacco and maize, Plant Cell 8:873-866).

phosphomannose isomerase (PMI): This segment next to the left border isthe mannose-6-phosphate isomerases gene from E. coli (Miles and Guest1984, Gene 32:41-48 ) fused to the SMAS promoter (Ni M, Cui D, EinsteinJ, Narasimhulu S, Vergara C E, Gelvin S B (1995) and Nos terminator. Thephosphomannose isomerase gene is used as a selection marker to selecttransgenic shoots on media containing D-mannose as the carbon source.

The components and sequence of pNOV2145 (FIG. 2) are set forth in SEQ IDNO:1. The components and sequence of pNOV2147 (FIG. 3) are set forth inSEQ ID NO:2.

EXAMPLE 2

Transformation and Regeneration

Mature dried soybean seeds (Var. S42 H1) were surface sterlized byreleasing chlorine gas inside a desiccator. Seeds were kept in petriplates and chlorine gas was produced by pouring 100 ml of Clorox® into abeaker and slowly adding 8 ml of concentrated HCl. Seeds were sterilizedby at least two gas release treatments each lasting for 8-18 hours.

Sterilized seeds (approximately 15-20 seeds per plate) were then placedon a germination medium containing 0.6% agar-solidified MS basal medium(Murashige and Skoog (1962) A revised medium for rapid growth andbioassays with tobacco callus cultures. Physiol Plant 15:473-479) and 2%sucrose. The pH was maintained at 5.8. The petri plates were placed in aroom at 37° C. for overnight growth or imbibition of seeds. The seedcoat was removed, followed by removing part of the hypocotyl, keepingabout 0.5 cm of the hypocotyl. One cotyledon was removed along with itsadjacent axillary shoot bud and was discarded. On the remainingcotyledon, the primary leaves were broken apart using a scalpel, leavingthe primary leaf bases on the epicotyl. (FIG. 4)

Agrobacterium strain (LBA 4404). containing the plasmid pNOV 2145(ZsGreen1and PMI, as described in Example 1) was streaked from frozenglycerol stocks onto YEP plates (yeast extract 10 g/L, peptone 5 g/L,NaCl 5g/L, bacto agar 15 g/L) containing appropriate antibiotic (100mg/L spectinomycin). Agrobacterium was then incubated at 27° C. for 1-2days. A scoop of Agrobacterium from plates were grown on 100 ml YEPliquid medium containing an antibiotic (100 mg/L spectinomycin) forovernight growth at 27° .C on a shaker. Bacterial suspensions werecentrifuged at about 1500 g for 15 minutes and resuspended to a densityof OD₆₆₀=0.2 or 0.65 in a co-cultivation liquid medium (B₅ salts 0.05X(Sigma), B₅ vitamins: (0.05X) (B₅ vitamin composition (1X): inositol 100mg/L, nicotinic acid 1 mg/L, pyridoxine HCl 1 mg/L, thiamine HCl 10mg/L), acetosyringone 40 mg/L, sucrose 20 g/L, IBAP 2 mg/L, GA₃ 0.25mg/L, MES (Morpholino ethanesulfonic acid) 3.9 g/L, and pH 5.4.

The explants containing the target tissue were immersed intoAgrobacterium suspension and incubated for 1-2 hours. The Agrobacteriumsuspension was poured off, and the treated explants were placed onto afilter paper inside co-cultivation plates. The adaxial side of theexplants was kept in contact with the filter paper. The co-cultivationsolid medium was composed of B₅ salts (Sigma, 0.05X), B₅ vitamins(0.05X), 40 mg/L acetosyringone, sucrose 20 g/L, BAP 2 mg/L, GA₃ 0.25mg/L, MES 3.9 g/L, and pH 5.4. The medium was solidified with 0.5%purified agar (Sigma).

The explants were co-cultivated with the Agrobacterium at 20-23° C. fora period of 2-5 days, under 16 h light/8 h dark conditions. Afterco-cultivation, the explants were washed in sterile water containing 250mg/L cefotaxime, primary and secondary meristems were removed, and theexplants were transferred to regeneration medium (i.e., REG-1 medium).During the regeneration process, any axillary shoots adjacent to thecotyledon were also removed to encourage growth from the area of theprimary leaf base.

REG-1 medium contained MS salts (1X), B₅ vitamins (1X), KNO₃ 1 g/L, BAP1 mg/L, ticarcillin 300 mg/L, cefotaxime 100 mg/L, glutamine 250 mg/L,asparagine 50 mg/L, mannose 15-30 g/L, sucrose 0, 0.25, and 1 g/L, pH5.6, and purified agar 10 g/L. Five explants were placed in each petriplate in an upright position, such that the epicotyl end of the explantwas inserted into the medium. The plates were kept inside a plasticcontainer and placed in a culture room at 22-25° C., under an 18-20 hrlight/4-6 hr dark cycle at 60-100 μE m⁻² S⁻¹. After 2 weeks on REG-1medium, explants were transferred to REG-2 medium, which contained MSsalts (1X) and B₅ vitamins (1X), KNO₃ 1 g/L, BAP 0.5 mg/L, ticarcillin300 mg/L, cefotaxime 100 mg/L, glutamine 250 mg/L, asparagine 50 mg/L,mannose 15 g/L, and sucrose 1 g/L. The media pH was maintained at 5.6,and the media was solidified with purified agar 10 g/L.

At 4-6 weeks, the soybean cultures were transferred to REG-3 medium forcontinuing selection and shoot development. REG-3 medium contained MSsalts (1X), B₅ vitamins (1X), KNO₃ 1 g/L, BAP 0.2 mg/L, GA₃ 0.5 mg/L,IBA 0.1 mg/L, ticarcillin 300 mg/L, cefotaxime 100 mg/L, glutamine 250mg/L, asparagine 50 mg/L, mannose 15 g/L, sucrose 1 g/L, pH 5.6, and themedium was solidified with purified agar 10 g/L. Dcad tissue was removedand explants with regenerating shoots were subcultured in fresh REG-3medium every two weeks. Elongated shoots were continuously harvestedfrom the cultures when they reached about 2-4 cm in length. At thattime, shoots were transferred to a rooting medium, which contained MSsalts (0.5X), B₅ Vitamins (0.5X), glutainine 250 mg/L, asparagine 50mg/L, KNO₃ 1 g/L, cefotaxime 100 mg/L, ticarcillin 300 mg/L, sucrose 15g/L, IBA 0.5 mg/L, pH 5.6, and purified agar 10 g/L.

Rooted transgenic shoots expressing a fluorescent protein gene(ZsGreen1) were transferred to 2″ pots which contained moistened Fafardgerminating mix (Conrad Fafard Inc., MA, USA) and were kept covered withplastic cups for maintaining moisture for approximately 2 weeks. Plantswere acclimatized at 27-29° C. day temperature, 21° C. nighttemperature, and a 16h photoperiod (20-40 μE m⁻² S⁻¹ light intensity).When new leaves began to emerge, plants were transferred to one-gallonpots which contained a soil mixture composed of 50-55% composted pinebark, 40-45% Peat, 5-10% Perlite (Sungrow Horticultural Supply, PineBluff, Ark.). Acclimatized soybean plants were grown in the greenhouseat 27-29° C. day temp, 21° C. night temp, 400-600 μE m⁻² S⁻¹ lightintensity, 70-95% relative humidity, and a 16 hr photoperiod. The plantswere fertilized with osmocote (Scotts-Sierra Horticultural ProductsCompany, Ohio; 17-6-12) twice (5-8 g/gallon soil) during the growthperiod. Transformation was confirmed by Taqman analysis for the presenceof the fluorescent protein gene as well as the PMI gene in the leaves ofthe greenhouse grown plants. Expression of the fluorescent protein genein the transformed soybean tissue was also confirmed by visualizing theexpression using a fluorescent microscope.

Six transgenic plants developed using the gene construct pNOV2145 wereconfirmed by Southern blot analyses. Progeny analysis of one event foreither the PMI gene or the ZsGreen1 gene revealed one integration siteof the T-DNA into the genome of the transformed soybean, and the progenysegregated in a 3:1 ratio in the T1 generation. TABLE 1 Transformedshoots expressing fluorescent protein gene (Zsgreen1) Transformed ExptNo. Gene construct shoots/explant Percent tr. shoots 75 pNOV2145 5/45 1189 pNOV2145 5/75 7

EXAMPLE 3

Soybean seeds (Var. S42 H1) were surface sterilized and explants wereprepared as described in Example 2.

Agrobacterium strain (LBA 4404) carrying the plasmid pNOV2147 wasprepared as described in Example 1. The final bacterial concentrationwas adjusted to OD ₆₆₀=0.60 with a co-cultivation liquid medium. Theconditions for explant preparation, Agrobacterium inoculation, andco-cultivation were the same as those described in Example 2.

Following three days of co-cultivation in a solid co-cultivation medium,excessive Agrobacterium was washed off, primary and secondary meristemswere removed, and the explants were transferred to REG-1 medium. Theywere cultured at 28-30° C. in 16h light and 8h dark conditions. After 2weeks on REG-1 medium, the cultures were transferred to REG-2 medium.During this regeneration process, only shoots arising from the base of aprimary leaf were kept. At about the 4th week, the shoot cultures weretransferred to REG-3 medium. They were then transferred to fresh REG-3medium every 10-14 days. As in REG-1 and REG-2 medium, only the newshoots arising from the base of a primary leaf were kept while the restof the shoots were removed. When elongated shoots reached about 24 cm inlength, they were separated from the rest of the shoot cultures andtransferred to rooting medium.

Five transgenic shoots out of 35 explants were identified as expressingthe cyano fluorescent protein gene (Table 2). TABLE 2 Transformed shootsexpressing the cyano fluorescent protein gene Experiment Transformed No.Gene construct shoots/explants % transformed shoots 92 pNOV2147 5/35 14

EXAMPLE 4

Mannose Treatment During Co-cultivation

The gene construct used in this example was pNOV2145 (which comprisesZsGreen1 and PMI genes, as described in Example 1). The procedures forpreparing the explants, Agrobacteria suspensions, and inoculation ofexplants with bacterial suspensions were carried out as described inExample 2. The final bacterial concentration was adjusted to OD ₆₆₀=0.55or 0.85.

Following the inoculation step, explants were transferred to aco-cultivation medium containing either 20 g/L sucrose or 15 g/L mannoseand were kept at 20-23° C. under 16h light and 8h dark conditions.

After 3-5 days of co-cultivation, expression of the fluorescent proteingene was visualized using a fluorescent microscope. Explants that wereinoculated with Agrobacterium in a mannose-containing co-cultivationmedium showed at least two-fold the number of fluorescent spots comparedto those co-cultivated in a sucrose-containing co-cultivation medium.Subsequent shoot regeneration and selection steps were followed as thosedescribed in Example 2.

A significant increase in the production of transformed shoots wasobserved in the experiments where mannose was included in theco-cultivation medium (Table 3). Five transformed shoots fromco-cultivation medium that included mannose were rooted and transferredto soil. Subsequent analysis by Taqman as well as Southern blotconfirmed the integration of the transgenes. Transgene expression in theT1 progeny confirmed the germline transmission of the transgenes. TABLE3 Transformed shoots expressing ZsGreen1 fluorescent protein gene whereexplants and Agrobacteria were co-cultivated in mannose or sucroseExperi- Transformed % ment Gene Co-culture in shoots/ Transfor- No.construct mannose/sucrose explants mation 87 pNOV2145 Sucrose 0/60 0Mannose 6/80 7.5 102 pNOV2145 Sucrose 1/20 5 Mannose 8/40 20

EXAMPLE 5

In this example, Agrobacterium EHA101 comprising the plasmid pNOV2105(SMAS-PMI SMAS-GUS, as described in Example 1) was used in soybeantransformation. The preparation of the explants, Agrobacteriasuspension, and inoculation of explants with Agrobacteria were the sameas those described in Example 2. The final bacterial concentration wasadjusted to OD ₆₆₀=0.45 or 0.6.

Following Agrobacterium inoculation, explants were transferred to aco-cultivation medium containing either 20 g/L sucrose or 15 g/Lmannose. Co-cultivation was carried out at 20-23° C. under a 16h lightand 8h dark conditions. Following 3-5 days of co-cultivation, GUS geneexpression was visualized using a histochemical gus assay. Explantsco-cultivated in mannose-containing co-cultivation medium showed atleast two-fold the number of GUS spots compared to those co-cultivatedin sucrose-containing co-cultivation medium. Shoot regeneration andselection were carried out as described in Example 2. A significantincrease in the production of transformed shoots was observed in theexperiment in which mannose was added into the co-cultivation medium.(Table 4). TABLE 4 Transformed shoots expressing GUS gene Experi-Transformed % ment Gene Co-cultivation in shoots/ Transfor- No.construct mannose/sucrose explant mation 63 pNOV2105 sucrose 5/60 8 81pNOV2105 Sucrose 2/30 7 Mannose 5/30 17

EXAMPLE 6

In this example, Agrobacterium EHA101 comprising the plasmid pBSC11234(FIG. 5) was used in soybean transformation. The components and sequenceof pBSC11234 are set forth in SEQ ID NO:3. pBSC11234 comprises a CMP-PMI: beta conglycinin-galactosidase gene construct. The preparation of theexplants, Agrobacteria suspension, and inoculation of explants withAgrobacteria were the same as those described in Example 2. The finalbacterial concentration was adjusted to OD ₆₆₀=0.6. The co-cultivationliquid medium contained B₅ salts (0.1X), B₅ vitamins (1X),acetosyringone 80 mg/L, sucrose 20 g/L, BAP 2 mg/L, GA₃ 0.25 mg/L, MES3.9 g/L, and pH 5.4. Solid co-cultivation medium was prepared byincorporating 5 g/L purified agar to the liquid co-cultivation medium.

Following Agrobacterium inoculation, explants were transferred to asolid co-cultivation medium and cultured at 20-24° C. under 16h lightand 8h dark conditions. Following 3-5 days of co-cultivation, primaryand secondary shoot meristems were removed and discarded, and theresulting explants were transferred to REG-4 medium, which contained B₅salts (1X), B₅ Vitamins (1X), BAP 1 mg/L, glutamine 50 mg/L, asparagine50 mg/L, cefotaxime 100 mg/L, ticarcillin 300 mg/L, mannose 15-20 g/L,sucrose 0, 0.25, or 1 g/L, purified agar 10 g/L, and pH at 5.6. After aperiod of 5-7 days, any shoot grown from the axillary meristem close tothe cotyledon was removed, and the explants were transferred to REG-5medium, which contained B₅ salts (1X), B₅ Vitamins (1X), BAP 0.5 mg/L,glutamine 50 mg/L, asparagine 50 mg/L, cefotaxime 100 mg/L, ticarcillin300 mg/L, mannose 15 g/L, sucrose 1 g/L, purified agar 10 g/L, and pH at5.6. At four weeks, explants were transferred to REG-6 medium forelongation of shoots. REG-6 medium contained MS salts (1X), MS Vitamins(1X) (MS vitamin composition: inositol 100 mg/L, nicotinic acid 0.5mg/L, pyridoxine HCl 0.5 mg/L, thiamine HCl 0.1 mg/L, glycine 2 mg/L),myo-inositol 200 mg/L, BAP 0.2 mg/L, zeatin riboside 0.5 mg/L, IBA 0.1mg/L, GA₃ 1 mg/L, glutamine 50 mg/L, asparagine 50 mg/L, ticarcillin 300mg/L, mannose 15 g/L, sucrose 5 g/L, silver nitrate 0.8 mg/L, purifiedagar 10 g/L, and pH 5.6. Explants were transferred to fresh REG-6 mediumevery two weeks. Elongated shoots (24 cm long) were removed and rootedin rooting medium and transferred to soil. The rooting medium containedMS salts (1X), B₅ Vitamins (1X), glutamine 100 mg/L, asparagine 100mg/L, IBA 0.7 mg/L, timentin 100 mg/L, and sucrose 15 g/L. Taqmananalysis confirmed the presence of the transgenes (alpha galactosidaseand phosphomannose isomerase) in leaf samples from two events.

EXAMPLE 7

In this example, Agrobacterium EHA101 comprising the plasmid pBSC11369(FIG. 6) was used in soybean transformation. The components and sequenceof pBSC11369 are set forth in SEQ ID NO:4. pBSC11369 comprises aCMP-HPT: CMP-ZsGreen1 gene construct. The preparation of the explants,Agrobacteria suspension, and inoculation of explants with Agrobacteriawere the same as those described in Example 2. The final bacterialconcentration was adjusted to OD ₆₆₀=0.6. The co-cultivation liquidmedium contained B₅ salts (0.1X), B₅ vitamins (1X), acetosyringone 80mg/L, sucrose 20 g/L, BAP 2 mg/L, GA₃ 0.25 mg/L, MES 3.9 g/L, and pH5.4. Solid co-cultivation medium was prepared by incorporating 5 g/Lpurified agar to the liquid co-cultivation medium.

Following Agrobacterium inoculation, explants wore transferred to asolid co-cultivation medium and cultured at 20-24° C. under 16h lightand 8h dark conditions. Following 3-5 days of co-cultivation, explantswere transferred to REG-7 medium after removing primary and secondarymeristems from the explants in order to encourage shoot growth from theprimary leaf base area REG-7 medium contained B₅ salts (1X), B₅ Vitamins(1X), BAP 1 mg/L, glutamine 50 mg/L, asparagine 50 mg/L, cefotaxime 100mg/L, ticarcillin 300 mg/L, sucrose 30 g/L, hygromycin 2-5 mg/L,purified agar 10 g/L, and pH 5.6. Explants were placed in an uprightposition such that the epicotyl end of the explant was inserted into themedium. After a period of 7-10 days, any shoots grown from the axillarymeristem close to the cotyledon were removed. Explants were transferredto fresh REG-8 medium, which contained B₅ salts (1X), B₅ Vitamins (1X),BAP 0.5 mg/L, glutamine 50 mg/L, asparagine 50 mg/L, cefotaxime 100mg/L, ticarcillin 300 mg/L, sucrose 30 g/L, purified agar 10 g/L, and pHat 5.6. After another two weeks, explants were transferred to REG-9medium and subcultured thereafter every two weeks. REG-9 mediumcontained MS salts (1X), MS Vitamins (1X), myo-inositol 200 mg/L, BAP0.2 mg/L, zeatin riboside 0.5 mg/L, IBA 0.1 mg/L, GA₃ 1 mg/L, glutamine50 mg/L, asparagine 50 mg/L, silver nitrate 0.8 mg/L, ticarcillin 300mg/L, sucrose 30 g/L, hygromycin 0.1-0.2 mg/L, purified agar 10 g/L, andpH 5.6. Elongated shoots (24 cm long) were removed, rooted in rootingmedium, and then transferred to soil. The rooting medium contained MSsalts (1X), B₅ Vitamins (1X), glutamine 100 mg/L, asparagine 100 mg/L,IBA 0.7 mg/L, timentin 100 mg/L, and sucrose 15 g/L. Taqman analysisconfirmed the presence of the transgenes (HPT as well as ZsGreen1) inleaf samples obtained from five events. Expression of the ZsGreen1 genein plant parts was confirmed by visualization under a fluorescentmicroscope.

All publications, patents, and patent applications cited herein areincorporated by reference. While in the foregoing specification thisinvention has been described in relation to certain preferredembodiments thereof, and many details have been set forth for purposesof illustration, it will be apparent to those skilled in the art thatthe invention is susceptible to additional embodiments and that certainof the details described herein may be varied considerably withoutdeparting from the basic principles of the invention.

1. A method for transforming soybean cells or tissue, comprising: (a)preparing an explant from a soybean seed by. (i) removing a hypocotylfrom said soybean seed; (ii) removing one cotyledon along with itsadjacent axillary bud, leaving primary leaves attached to a remainingcotyledon; and (iii) removing a portion of a primary leaf from saidremaining cotyledon, thereby generating a primary leaf base; and (b)co-cultivating said explant with Agrobacterium comprising at least onenucleic acid of interest to be incorporated into a genome of one or moresoybean cells.
 2. The method of claim 1, further comprising cultivatingat least one formed shoot in a medium containing a selection agent. 3.The method of claim 2, wherein said at least one nucleic acid ofinterest comprises a selectable marker gene.
 4. The method of claim 3,wherein said selectable marker gene is a phosphomannose isomerase gene.5. The method of claim 4, wherein said selection agent is mannose. 6.The method of claim 4, wherein co-cultivation with said Agrobacterium iscarried out in the presence of mannose.
 7. The method of claim 2,further comprising inducing shoot formation from said primary leaf base.8. The method of claim 7, wherein shoot formation is induced byculturing said primary leaf base in a medium comprising a shoot-inducinghormone.
 9. The method of claim 8, wherein said shoot-inducing hormonecomprises at least one of an auxin, a cytokinin, and a gibberellic acid.10. The method of claim 9, wherein said auxin is selected from the groupconsisting of IAA, NAA, and IBA.
 11. The method of claim 9, wherein saidcytokinin is selected from the group consisting of benzylaminopurine(BAP), thidiazuron, kinetin, and isopentenyl adenine.
 12. The method ofclaim 7, wherein induction of shoot formation comprises removing one ormore of a primary meristem, a secondary meristem, and an axillarymeristem attached to a cotyledon.
 13. The method of claim 7, furthercomprising selecting a transformed shoot.
 14. The method of claim 13,further comprising regenerating a selected transformed shoot into asoybean plant.
 15. The method of claim 1, wherein said soybean seed is amature seed.
 16. The method of claim 1, wherein said soybean seed is animmature seed.
 17. The method of claim 1, wherein said soybean seed is agerminated seed.
 18. A method for producing a stably transformed soybeanplant, comprising: (a) preparing an explant from a soybean seed by: (i)removing a hypocotyl from said soybean seed; (ii) removing one cotyledonalong with its adjacent axillary bud, leaving primary leaves attached toa remaining cotyledon; and (iii) removing a portion of each primary leaffrom said remaining cotyledon, thereby generating a pair of primary leafbases; (b) co-cultivating said explant with Agrobacterium comprising anucleic acid of interest to be incorporated into a genome of a soybeancell; (c) inducing shoot formation from each primary leaf base; (d)cultivating at least one formed shoot in a medium containing a selectionagent; (e) selecting a transformed shoot; and (f) regenerating aselected transformed shoot into a soybean plant.
 19. A transgenicsoybean plant regenerated from soybean cells or tissue transformedaccording to the method of claim
 1. 20. A transgenic seed produced bythe transgenic plant of claim
 19. 21. A transgenic soybean plantregenerated from soybean cells or tissue transformed according to themethod of claim
 18. 22. A transgenic seed produced by the transgenicplant of claim 21.